Understanding the rules by which variation in primary DNA sequence impacts variation for quantitative traits in natural populations is critical for predicting responses to natural and artiicial selection as well as disease risk in human populations. Establishing unambiguous causality for SNPs associated with phenotypic variation is hampered in human genetic studies because of large LD blocks. Furthermore, one major question in genetics is to what extent naturally occurring alternative alleles associated with phenotypic variation differentially affect expression of ensembles of genes. This issue of indirect effects of alternative alleles on network structure i of paramount importance for the interpretation of human GWA studies and can be addressed in the Drosophila melanogaster model. This application seeks to continue our studies on the genetic architecture of olfactory behavior in Drosophila melanogaster as a model quantitative trait. Chemical signals are essential for survival and reproduction, providing the stage on which evolutionary forces can act. Chemosensation is essential for food localization, avoidance of toxins or predators, and assessment of mating partners and oviposition sites. Chemical signals also drive aggression, courtship and, in mammals, affiliative behaviors and parental care. Furthermore, olfactory deficits in people can compromise quality of life and, consequently, adversely affect nutrition - especially in the elderly - and olfactory decline can be an early indication of neurodegenerative disease. A long-standing problem in olfaction, however, is our incomplete understanding of the causes of individual variation in olfactory perception. Drosophila provides an excellent system for studies on the genetic basis of variation in olfactory behavior, since it has one of the best characterized olfactory systems and large numbers of genetically identical individuals can be grown under controlled environmental conditions rapidly, economically, and without regulatory restrictions, allowing exquisite control over both the genetic background and the environment. In addition, the Drosophila melanogaster Genetic Reference Panel (DGRP), which consists of inbred wild-derived lines with fully sequenced genomes, enables us to capitalize on natural variation to investigate the genetic underpinnings of variation in complex traits. The DGRP represents a reference population of replicate genotypes in which all molecular variants are known and has enabled us to perform the first genome-wide association (GWA) studies for olfactory behavior in Drosophila. Building on advances from the previous project period, the major thrust of the continuation of our research program is to provide a model blueprint for advancing GWA analyses from the single gene level to the level of network associations and to probe how altered expression levels of genes within the network or SNPs that affect phenotypic variation affect the underlying trait-associated genetic network structure. We will accomplish this through the following specific aims: Specific Aim 1a - Assessing the effects of targeted RNAi knockdown on olfactory behavior with cell- specific drivers; Specific Aim 1b - Combining targeted RNAi knockdown and genome-wide transcriptional profiling to expand the network, while assessing network connectivity; Specific Aim 2 - Assessing the effects of alternative alleles in co-isogenic null mutant backgrounds on olfactory behavior; and, Specific Aim 3 - Assessing the effects of alternative alleles in co-isogenic null mutant backgrounds on the organization of transcriptional networks. Results from these studies will not only set a new standard for Drosophila GWA analyses, but provide conceptual advances that can be generalized to other complex traits, not only in Drosophila, but across phyla.